Method and apparatus for measuring protein post-translational modification

ABSTRACT

The present invention includes a method for analyzing reactions. The method includes the steps of providing a solution of at least one acceptor chemical and at least one donor chemical. The donor chemical is capable of donating a chemical moiety to the acceptor chemical. The solution further includes at least one controller chemical that affects the reaction between the donor chemical and the acceptor chemical. The solution is then incubated so that a portion of the acceptor chemical reacts with the donor chemical form an acceptor product. Unreacted donor chemical is separated from the acceptor product. The acceptor product or the donor chemical is then measured using X-ray fluorescence. Another aspect of the present invention includes a method for analyzing protein function. The method includes the steps of providing a solution of at least one acceptor chemical and at least one donor chemical. The donor chemical is capable of donating a chemical moiety to the acceptor chemical. The donor chemical includes a functional group selected from ester, anhydride, imide, acyl halide, and amide. The solution is then incubated so that a portion of the acceptor chemical reacts with the donor chemical to form an acceptor product. Unreacted donor chemical is separated from the acceptor product. The acceptor product or the donor chemical is then measured using X-ray fluorescence. Yet another aspect of the present invention includes a method for analyzing protein function. The method includes the steps of providing a solution of at least one acceptor chemical and at least one donor chemical. The solution is then incubated so that a portion of the acceptor chemical reacts with the donor chemical to form an acceptor product. Unreacted donor chemical is separated from the acceptor product. The acceptor product or the donor chemical is then measured using X-ray fluorescence. An additional analytical method is also used to measure either the acceptor product or the donor chemical.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/239,459, filed Sep. 26, 2008, which claims the priority of U.S.Provisional Patent Application 60/995,997 entitled “Method and Apparatusfor Measuring Protein Post Translational Modification,” which was filedon Sep. 28, 2007, incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the analysis of protein function.

BACKGROUND OF THE INVENTION

Post-translational modifications are chemical changes to proteins thatoccur after the primary structure of the protein has been completed viatranslation. Post-translational modifications include, but are notlimited to, phosphorylation, dephosphorylation, proteolysis, proteinligation, glycosylation, sulfation, methylation, and ubiquitination.

Post-translational modifications influence protein behavior. Forexample, insulin is formed by the post-translational modification ofproinsulin, which itself is formed by the post-translationalmodification of preproinsulin. The post-translational addition orremoval of phosphorus from proteins plays a regulatory role in manybiochemical pathways and signal transduction pathways.

The analysis of post-translational modifications often requires laborintensive sample preparation and expensive or hazardous chemicalreagents such as radioactive materials. For example, protein kinaseassays often include the use of radioactively labeled ATP as phosphatedonor to a substrate peptide or protein. Following the kinase reaction,the substrate is separated from unreacted radioactive ATP. Anyradioactivity incorporated into the substrate is measured, such as byscintillation counting. This assay has drawbacks. The assay generatesradioactive waste. Radioactive phosphorus has a short half life, sofresh reagent must be frequently acquired. The assay requires at leastmicromolar concentrations of ATP, which is one thousand times greaterthan the millimolar biological concentration of ATP. The concentrationof substrate in the assay is often much higher than expected substrateconcentrations in vivo.

There remains a need for simpler methods for measuringpost-translational modification. The present invention is designed toaddress that need.

SUMMARY OF THE PRESENT INVENTION

One aspect of the present invention includes a method for analyzingreactions. The method includes the steps of providing a solution of atleast one acceptor chemical and at least one donor chemical. The donorchemical is capable of donating a chemical moiety to the acceptorchemical. The solution further includes at least one controller chemicalthat affects the reaction between the donor chemical and the acceptorchemical. The solution is then incubated so that a portion of theacceptor chemical reacts with the donor chemical to form an acceptorproduct. Unreacted donor chemical is separated from the acceptorproduct. The acceptor product or the donor chemical is then measuredusing X-ray fluorescence.

Another aspect of the present invention includes a method for analyzingprotein function. The method includes the steps of providing a solutionof at least one acceptor chemical and at least one donor chemical. Thedonor chemical is capable of donating a chemical moiety to the acceptorchemical. The donor chemical includes a functional group selected fromester, anhydride, imide, acyl halide, and amide. The solution is thenincubated so that a portion of the acceptor chemical reacts with thedonor chemical to form an acceptor product. Unreacted donor chemical isseparated from the acceptor product. The acceptor product or the donorchemical is then measured using X-ray fluorescence.

Yet another aspect of the present invention includes a method foranalyzing protein function. The method includes the steps of providing asolution of at least one acceptor chemical and at least one donorchemical. The solution is then incubated so that a portion of theacceptor chemical reacts with the donor chemical to form an acceptorproduct. Unreacted donor chemical is separated from the acceptorproduct. The acceptor product or the donor chemical is then measuredusing X-ray fluorescence. An additional analytical method is also usedto measure either the acceptor product or the donor chemical.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

FIGURES

FIG. 1 shows a flowchart depicting the steps in the method of thepresent invention.

FIG. 2 shows a flowchart depicting the steps in another method of thepresent invention.

FIG. 3 shows data obtained by using the method of the present inventionas described in Example 2.

DETAILED DESCRIPTION

An embodiment of the present invention is a method to determine theeffect of chemicals on proteins and protein function, as depicted in theflowchart in FIG. 1. The invention comprises the steps of providing asolution (FIG. 1, Box 1), incubating the solution, and measuring thesolution. The solution comprises a solvent, an acceptor chemical, adonor chemical, and a controller chemical. The solution is incubated fora period of time sufficient for a portion of the donor chemical to reactwith a portion the acceptor chemical to form a donor product and anacceptor product (FIG. 1, Box 2). The donated chemical element can bedonated alone or as part of a chemical moiety or functional group. Anyunreacted donor chemical is then separated from the acceptor product(FIG. 1, Box 3). Either the unreacted donor chemical or the acceptorproduct is then measured using X-ray fluorescence (FIG. 1, Box 4).

A second embodiment of the present invention is a method to determinethe activity of proteins. The second embodiment comprises the steps ofproviding a solution comprising at least one acceptor chemical and atleast one donor chemical (FIG. 1, Box 1). The donor chemical is capableof donating a chemical element to the acceptor chemical to form anacceptor product. The donated chemical element can be donated alone oras part of a chemical moiety or functional group. In the secondembodiment, the donor comprises a functional group selected from ester,anhydride, amide, and combinations thereof. The solution is thenincubated to allow at least a portion of the acceptor chemical to reactwith the donor chemical to form a product chemical (FIG. 1, Box 2). Thedonor chemical and the acceptor product are then separated (FIG. 1, Box3). The donor chemical or the acceptor product is then measured usingX-ray fluorescence (FIG. 1, Box 4). The solution of the secondembodiment of the present invention preferably also comprises at leastone controller chemical.

A third embodiment of the present invention is a method to determine theactivity of proteins, as depicted in FIG. 2. The third embodimentcomprises the steps of providing a solution comprising at least oneacceptor chemical and at least one donor chemical (FIG. 2, Box 1). Thedonor chemical is capable of donating a chemical element to the acceptorchemical to form an acceptor product. The donated chemical element canbe donated alone or as part of a chemical moiety or functional group.The solution is incubated to allow a portion of the acceptor chemical toreact with the donor chemical to form a product chemical (FIG. 2, Box2). Unreacted donor chemical is then separated from the acceptor product(FIG. 2, Box 3). The donor chemical or the acceptor product is thenmeasured using X-ray fluorescence (FIG. 2, Box 4). The chemical that ismeasured using X-ray fluorescence is also quantified using a secondmeasurement technique (FIG. 2, Box 4).

The solution comprises a solvent, an acceptor chemical, and a donorchemical. The solvent preferably does not contain at least one of theelements selected from the list phosphorus, sulfur and chlorine. Thesolvent is more preferably water, and most preferably the solvent isbuffered water.

The solution is allowed to incubate for a reaction to occur. In thepresent invention, incubate refers to allowing the reaction to proceedat any temperature.

Many chemicals require a buffer to maintain the pH within a particularrange (e.g. a pH buffer), or to maintain the redox state of a chemical(e.g. a redox buffer), or to maintain an ionic strength (e.g. anisotonic buffer). Many buffers contain elements which might interferewith the measurement of the chemical. The buffer should preferably befree of at least one chemical element having an atomic number of greaterthan four, where that chemical element is present in the donor chemicalor acceptor molecule. The buffer should more preferably be free of atleast one chemical element having an atomic number of greater thaneight, where that chemical element is present in the donor chemical oracceptor product. The buffer should preferably be free of at least oneof the following chemicals or functional groups: dimethylsulfoxide,thiols, sulfate anion, sulfonate anions, chloride anion, bromide anion,fluoride anion, iodide anion, perchlorate anion, phosphate anion, andphosphonate anions. The buffer preferably comprises one or more of thefollowing chemical or functional groups: amine, imine, nitrate anion,nitrite anion, ammonium cation, acetate anion, carboxylate anion,conjugate bases of carboxylic acids, carbonate anion, and iminiumcation; these chemicals offer the correct buffering properties withminimal X-ray fluorescence interference.

The solution may be redox buffered using chemicals such asdithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). However,it is highly preferable that the substrate is separated from anyphosphorus or sulfur in the redox buffer. Preferred redox buffers do notcontain phosphorus or sulfur, or else they are solid supported such asImmobilized TCEP Disulfide Reducing Gel, which is available from ThermoFisher Scientific Inc. PO Box 117, Rockford, Ill. 61105 USA.

The donor chemical comprises an element having an atomic number ofgreater than eight. The donor chemical reacts with the acceptor chemicalto transfer this chemical element having an atomic number greater thaneight, either by itself or as part of a larger chemical group, to theacceptor chemical to form an acceptor product. The element to be donatedis preferably selected from the list sulfur, phosphorus, selenium,chlorine, bromine, iodine, functional groups comprising at least one ofthese chemical elements, and combinations thereof. After the donorchemical has reacted to transfer the chemical element having an atomicnumber greater than eight, the donor chemical is called the donorproduct. The donor chemical preferably comprises at least one of thechemical functional groups selected from the list of anhydride, ester,amide, imide, acyl halides, and combinations thereof. Functional groupsinclude those not based on carbon, such as anhydrides or phosphoricacid, phosphonic acid, their sulfur analogs, and the like are consideredto be anhydrides; esters such as phosphoric esters and phosphonic estersand their sulfur analogs are considered to be esters; amides such asphosphoric amides and phosphonic amides and their sulfur analogs and thelike are considered to be amides; imides such as phosphoric imides andphosphonic imides and their sulfur analogs and the like are consideredto be imides; acyl halides such as phosphoryl halides and phosphonylhalides and their sulfur analogs and the like are considered to be acylhalides. Examples of donor chemicals are adenosine triphosphate, inosinetriphosphate, guanosine-5′-triphosphate, and3′-phosphoadenosine-5′-phosphosulfate. The donor chemical preferably hasa concentration of less than about 100 millimolar.

The acceptor chemical reacts with the donor chemical to accept achemical element having a chemical element having an atomic numbergreater than eight, either by itself or as part of a larger chemicalgroup, from the donor chemical. The acceptor chemical preferably alsocomprises at least one element having an atomic number greater thaneight that is not the same as the element donated by the donor chemical;if this is the case, then the efficiency of the reaction between thedonor chemical and the acceptor chemical may be calculated from themeasured values of these two elements. For example, if the acceptorchemical comprises a sulfur atom, and the donated functional groupcomprises a phosphorus atom, then the ratio of the phosphorus X-rayfluorescence signal to the sulfur X-ray fluorescence signal allows thecompleteness of the reaction to be measured with a single measurementtechnique. The acceptor chemical preferably contains at least one of thefunctional groups R—NH₂, RN(X)H, R—O—H, R—O⁻, R—S—H, R—S⁻ where R iscarbon or hydrogen, and X may be any chemical element, R—O⁻ refers to analkoxide anion or aryloxide anion, and R—S⁻ refers to an alkyl or arylthiolate anion. Examples of acceptor chemicals are proteins, aminoacids, peptides, polymers comprising amino acids, oligomers comprisingamino acids, nucleotides, polymers comprising nucleotides, oligomerscomprising nucleotides, and water. The acceptor chemical preferably hasa concentration of less than about 100 millimolar.

The reaction between the donor chemical and the acceptor chemicalpreferably forms or breaks at least one covalent bond.

The solution comprises one or more controller chemicals. The controllerchemicals may accelerate the rate of the reaction between the donorchemical and the acceptor chemical. The controller chemicals maydecelerate the rate of the reaction between the donor chemical and theacceptor chemical. The controller chemicals may increase or decrease theturnover number of a catalyst. Different controller chemicals in thesame solution may accelerate or decelerate the reaction between theacceptor chemical and the donor chemical by various amounts. Examples ofcontroller chemicals are adenosine diphosphate;phthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine;1-(5-Isoquinolinesulfonyl)-1H-hexahydro-1,4-diazepine;1-(5-Isoquinolinesulfonyl)-2-methylpiperazine;1-(5-Isoquinolinesulfonyl)-piperazine;1,2-Dimethoxy-N-methyl(1,3)benzodioxolo(5,6-c)phenanthridiniumchloride;1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine;2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one;2,3-Dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide;2,5-Dihydroxymethylcinnamate;2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide;3-[[4-(dimethylamino)phenyl]methylene]-1,3-dihydro-2H-indol-2-one;4-(2,5-Dihydroxy-benzylamino)benzoic Acid Adamantan-1-yl Ester;4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline; 4,5,6,7-Tetrabromo-2-azabenzimidazole;4-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline;4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,d]pyrimidine;4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;4-Amino-5-iodo-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidine;4′-Amino-6-Hydroxyflavone; 4-Amino-N-(2,5-dihydroxybenzyl)adamantylBenzoate; 4-Amino-N-(2,5-dihydroxybenzyl)methyl benzoate;5,6-Dichloro-1-b-D-ribofuranosyl benzimidazole;5,7-Dimethoxy-3-(4-pyridinyl)quinoline;6-Amino-4-methyl-8-(β-D-ribofuranosyl)4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine;Bisindolylmaleimide IX; N-(2′-Guanidinoethyl)-5-isoquinolinesulfonamide;N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide;N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide;N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide;N-Benzyl-3,4-dihydroxy-benzylidenecyanoacetamide; Picropodophyllin;Quercetin; rac-2-methyl-1-octadecyl-glycero-(3)-phosphocholine;Rapamycin; Rottlerin; Staurosporine;trans-3,3′,4,5′-Tetrahydroxystilbene,4-[(1E)-2-(3,5-Dihydroxyphenyl)ethenyl]-1,2-benzenediol;trans-4-[(1R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide;wortmannin; metal; enzyme inhibitors; protein inhibitors; catalysts;enzymes; proteins; amino acids; peptides; polymers comprising peptides,carbohydrates; lipids; nucleotides; polymers comprising nucleotides;reactive oxygen species; and reactive nitrogen species. The solutionpreferably includes at least two controller chemicals. The solutionpreferably comprises at least one controller chemical that increases therate of reaction between the donor chemical and the acceptor chemicaland at least one controller chemical that decreases the rate of reactionbetween the donor chemical and the acceptor chemical. The solution mostpreferably includes at least one enzyme; more preferably the solutionincludes a metal that enhances the activity of this enzyme; and mostpreferably the solution also includes at least one chemical thatinhibits the activity of this enzyme.

The donor chemical, acceptor chemical, controller chemical, orcombinations thereof may be chemically attached to a surface orotherwise immobilized.

The donor chemical, acceptor chemical, solvent, or controller chemicalsmay be derived from biological specimens such as cell cultures, tissuesamples, tissue cultures, biopsy samples, blood samples, and the like.

After the solution is allowed to incubate for a period of timesufficient for a portion of the donor chemical to react with a portionof the acceptor chemical, the donor chemical is then separated from theacceptor chemical and acceptor product. The entire solution may besubjected to this separation. Alternatively, a portion of the solutionmay be subjected to this separation. If a portion of the solution issubjected to a separation, then a kinetic analysis of the reaction maybe performed by comparing the amount of the reaction between the donorchemical and the acceptor chemical which has occurred at various times.Other ways to perform kinetic analyses include varying theconcentrations of the donor chemical, the acceptor chemical, and anycontroller chemicals. These concentrations may be varied together orindependently.

The separation may be conveniently performed using chromatography, suchas gel filtration chromatography or size exclusion chromatography, suchas a Quick Spin Protein Column using Sephadex G-25, available from RocheApplied Science, PO Box 50414, Indianapolis, Ind., 46250. Thisseparation is amenable to multiplexing using a well plate format, suchas a 96-well, 384-well, or 1536-well plate format. Separations systemssuch as Zeba 96-well plates available from Pierce Biotechnology Inc., POBox 117, Rockford, Ill., 61105, are particularly convenient. Separationmay be expedited using a centrifuge, such as a the IEC CL40 availablefrom Thermo Fisher Scientific, product #11210923, 450 Fortune Blvd,Milford, Mass., 01757; or a vacuum manifold, such as a Vacuum apparatussuch as the MultiScreen Vacuum manifold with Direct Stack fromMillipore, 290 Concord Road, Billerica, Mass. 01821, attached to astandard vacuum pump (for example, Millipore, Catalog #WP61 115 60) alsoavailable from Millipore. An alternative method of separation isultrafiltration, such as might be performed using a Centricon YM-3centrifuged for 3 hours at 7000 g. Other alternatives includeextraction, selective precipitation, and immunoprecipitation. The keycharacteristic of the separation is that acceptor product and unreacteddonor chemical are separated. Separation using a centrifuge isespecially convenient, because it allows the use of well plates.

To facilitate the separation, the donor chemical and the acceptorproduct preferably differ in their molecular weights by a factor of atleast 10, and more preferably by a factor of at least 20. Thisdifference in molecular weights will allow convenient separation byultrafiltration and gel filtration chromatography. Alternatively, thedonor chemical and the acceptor product may preferably have differentionic charges at the same pH value. This difference in ionic stateallows convenient separation using anion exchange chromatography.Alternatively, the donor chemical and the acceptor product havewater-octanol partitioning coefficients that differ by a factor of atleast 10, and more preferably by a factor of at least 100. Thisdifference in water-octanol partitioning coefficients allows convenientseparation by extraction or many chromatographic methods. The separationpreferably separates the donor chemical from the acceptor chemical andthe acceptor product.

After the donor chemical and the acceptor product are separated, eitheror both the donor chemical and the acceptor product are measured usingx-ray fluorescence. The measurement may be conveniently obtained usingan X-ray fluorescence spectrometer. An XRF spectrometer is an apparatuscapable of irradiating a sample with an X-ray beam, detecting the X-rayfluorescence from the sample, and using the X-ray fluorescence todetermine which elements are present in the sample and measuring thequantity of these elements. The X-ray fluorescence measurement may beobtained using the EDAX Eagle XPL energy dispersive X-ray fluorescencespectrometer, equipped with a microfocus X-ray tube, lithium driftedsilicon solid-state detector, processing electronics, and vendorsupplied operating software, available from the EDAX division of Ametek,91 McKee Drive Mahwah, N.J. 07430. The X-ray fluorescence measurementmay be obtained using the ZSX Primus, available from Rigaku Americas,9009 New Trails Drive, The Woodlands, Tex. 77381. The x-ray fluorescenceinstrument preferably comprises at least one of the following: amonocapillary focusing optic, polycapillary focusing optic, acollimator, a microfocus X-ray tube, a synchrotron X-ray source, alinear accelerator X-ray source, a rhodium X-ray tube, a molybdenumX-ray tube, a chromium X-ray tube, a silver X-ray tube, a palladiumX-ray tube, a monochromatic X-ray source, a polychromatic X-ray source,a polarized X-ray source, a confocal X-ray fluorescence spectrometerfocusing arrangement, a PIN diode detector, a semiconductor X-raydetector, a germanium or doped germanium X-ray detector, a silicon ordoped silicon X-ray detector, a wavelength dispersive X-ray fluorescencespectrometer, an energy dispersive X-ray fluorescence spectrometer,total reflectance X-ray fluorescence spectrometer, and the like.Preferably, the x-ray excitation source emits x-ray having apolychromatic x-ray excitation spectrum, and more preferably the x-rayexcitation source emits x-rays having a spectrum with at least twomaxima. Excitation with polychromatic x-rays increases the efficiencyfor exciting more than one chemical element in the sample beinganalyzed, or for exciting more than one spectral feature in the chemicalanalyte being analyzed.

The sample of donor chemical or acceptor product being measuredpreferably has at least a portion of the solvent removed. Preferably atleast 80% of the solvent is removed; more preferably substantially allof the solvent is removed. Evaporation is a convenient method forremoving the solvent. The solvent is preferably evaporated usingelevated temperatures that are above about 22° C. or reduced atmosphericpressure that is below about 760 torr. The solvent may be convenientlyremoved using a vacuum centrifuge, such as a Savant Speed Vac Plus SC250DDA or a Thermo Savant SPD 1010 SpeedVac®.

The sample of donor chemical or acceptor product is preferablyconcentrated into an area which is comparable in size to the x-rayexcitation beam. The area containing more than about 50% of the mass ofthe sample to be measured should have an area which is within a factorof 100 of the area containing more than about 50% of the x-rays in thex-ray excitation beam. Preferably, the area containing more than about50% of the sample has an area of less than about 0.005 squarecentimeters. If the area of x-ray excitation beam as it illuminates thesample is significantly greater than the area of the sample, then x-rayphotons are wasted. If the area of x-ray excitation beam as itilluminates the sample is significantly smaller than the area of thesample, then the measurement time will be unnecessarily long or else aportion of the sample will be wasted by its not being measured.

If the solvent containing the sample is evaporated, the dried orpartially dried sample of acceptor product or donor chemical ispreferably deposited onto a deposition substrate. The depositionsubstrate is preferably substantially free of at least one of theelements sulfur, phosphorus, or chlorine. Examples of suitable materialsfor the deposition substrate include Porvair #229302, Porvair #229112,Porvair #229058, Porvair #229304, and Porvair #229301, all of which areavailable from Porvair plc, Brampton House, 50 Bergen Way, King's Lynn,Norfolk PE30 2JG, U.K.), aluminum foils (examples: Microseal ‘F’ Foilfrom Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, Calif.94547). Other materials that may be used for the deposition substrateinclude: Super-Thin Polyester Surface-Protection Tape,Chemical-Resistant Surlyn Surface-Protection Tape, Abrasion-ResistantPolyurethane Surface-Protection Tape, Heat-Resistant Kapton Tape withSilicone Adhesive or with Acrylic Adhesive, UV-Resistant PolyethyleneSurface-Protection Tape, Clean-Release Polyethylene Surface-ProtectionTape, Low-Static Polyimide Tape, all available from McMaster-Carr, 6100Fulton Industrial Blvd., Atlanta, Ga. 30336-2852. Other materials whichmay be used for the deposition substrate include polypropylene,available from Lebow Company, 5960 Mandarin Ave., Goleta, Calif. 93117U.S.A. Other substrates that are conveniently used for the depositionsubstrate include AP1, AP3, ProLINE Series 10, ProLINE Series 20,DuraBeryllium substrates from Moxtek, 452 West 1260 North, Orem, Utah84057. Other materials which may be used for the deposition substrateinclude Ultralene®, mylar, polycarbonate, prolene, and kapton, availablefrom SPEX CertiPrep Ltd, 2 Dalston Gardens, Stanmore, Middlesex HA7 1BQ,ENGLAND. Other materials that may be used as the deposition substrateinclude Hostaphan®, polyester, and Etnom® available from ChemplexIndustries, Inc., 2820 SW 42nd Avenue, Palm City, Fla. 34990-5573 USA.Another material that may be conveniently used is Zone Free Film PartZAF-PE-50, available from Excel Scientific, 18350 George Blvd,Victorville, Calif., 92394. Other useful substrates are glass andsilicon. This list is not exhaustive, and other materials may be used asthe deposition substrate. The deposition substrate is also preferablysubstantially free of elements which have X-ray fluorescence emissionpeaks having energies of between 1.9 KeV and 3 KeV, because these peakstend to interfere with the signals of most interest to biochemical andbiological applications. Elements which have X-Ray Fluorescence emissionpeaks having energies of between 1.9 KeV and 3 KeV are: osmium, yttrium,iridium, phosphorus, zirconium, platinum, gold, niobium, mercury,thallium, molybdenum, sulfur, lead, bismuth, technetium, ruthenium,chlorine, rhodium, palladium, argon, silver, and thorium. If an x-rayfluorescence spectrometer is used which uses an x-ray detector whichcomprises silicon, then the deposition substrate is also preferably freeof elements which have X-Ray Fluorescence escape peaks (i.e. x-rayfluorescence emission peaks minus 1.74 KeV) having energies of between1.9 KeV and 3 KeV, because these escape peaks tend to interfere with thesignals of most interest for biochemical and biological applications.Elements which have X-Ray Fluorescence escape peaks having energies ofbetween 1.9 KeV and 3 KeV are: calcium, tellurium, iodine, scandium,xenon, cesium, barium, titanium, and lanthanum. “Substantially free” isdefined herein as being less than about 4% by weight. The depositionsubstrate may have additional chemical elements, which may be used formeasuring the thickness of the sample. If wavelength dispersive x-rayfluorescence is used, then the elemental purity of the depositionsubstrate is not as important; in this case, the film should besubstantially free of the element or elements which are being used toquantify the sample. The deposition substrate may be treated to increaseprotein adhesion; a non-inclusive list of treatments includes treatingthe deposition substrate with oxygen or nitrogen plasma or withpoly-lysine.

The chemical measured using x-ray fluorescence may be measured using oneor more second analytical techniques. Examples of second analyticaltechniques include the addition of standard or internal standard thatmay be measured using x-ray fluorescence; other spectrometric techniquesthan x-ray fluorescence such as radiometric analysis or addedradioactive materials, ultraviolet or visible spectrometry, infraredspectrometry, surface plasmon resonance, nuclear magnetic resonancespectrometry, terahertz spectrometry, microwave spectrometry, surfaceplasmon resonance spectrometry, mass spectrometry. An example of asecond analytical technique is the Quick Start Bradford Protein Assay,available from Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr.,Hercules, Calif. 94547 USA, and performed according to manufacturer'sinstructions. Any of these techniques may be used with or without addedstandards, dyes, tracers, and the like.

EXAMPLES Example 1

NAD+ kinase served as the acceptor chemical and as a controllerchemical. Adenosine triphosphate served as the donor chemical. Magnesiumchloride and acetic acid served as controller chemicals. PhosphorylatedNAD kinase served as the acceptor product. Adenosine diphosphate servedas the donor product. A solution was prepared by combining 100microliters of a solution of NAD kinase [880 micromolar] in water with a200 microliters of a solution of adenosine triphosphate [5 millimolar],magnesium chloride [10 millimolar], tris(hydroxymethyl)aminomethanebuffer [100 millimolar, pH 7.5] in water (FIG. 1, Box 1). The reactionwas allowed to incubate for 10 seconds, after which 200 microliters of asolution of 5% acetic acid in water was added (FIG. 1, Box 2). Theacceptor product was separated from the adenosine triphosphate andadenosine diphosphate using a Microcon 3000 molecular weight cut off(MWCO) ultrafilter, available from Millipore Corporate Headquarters, 290Concord Road, Billerica, Mass. 01821, USA (FIG. 1, Box 3). The NADP wasmeasured using an x-ray fluorescence spectrometer equipped with a 50watt chromium anode x-ray tube and a silicon drift detector (FIG. 1, Box4). The ratio of the phosphorus x-ray fluorescence signal to sulfurx-ray fluorescence signal was 0.030501 for the NAD, and 0.264657 for thephosphorylated NAD.

Example 2

A set of peptides having the formula: polystyrenebead-LINKER-cysteine₁-xxx₁-xxx₂-xxx₃-xxx₄-cysteine₂, where xxx₁, xxx₂,xxx₃, and xxx₄ are independently selected from the amino acids {alanine,arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine} served as the donorchemical. “Polystyrene bead-LINKER” refers to AM resin such as availablefrom Rapp Polymere GmbH, Ernst-Simon-Str. 9, D 72072 Tübingen, Germany.Water served as the acceptor chemical. Trypsin and K₂PtCl₄ served ascontroller chemicals. The peptides having the formula: polystyrenebead-LINKER-cysteine₁-xxx₁-xxx₂-xxx₃-xxx₄-CO₂H served as the donorproduct, where xxx₁, xxx₂, xxx₃, and xxx₄ are independently selectedfrom the amino acids {alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine,valine, and no amino acid} served as the donor chemical. The peptideshaving the formula: polystyrene bead: H₂N-xxx₁-xxx₂-xxx₃-xxx₄-cysteineserved as the donor product, where xxx₁, xxx₂, xxx₃, and xxx₄ areindependently selected from the amino acids {alanine, arginine,asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, and no amino acid} served as the acceptorproduct. A 250 microliter solution was prepared of resin bound peptides[in suspension] and K₂PtCl₄ [50 mM]; this solution was incubated for 4hours, after which 500 microliters of porcine trypsin [45 micromolar]and ammonium bicarbonate [40 millimolar] was added (FIG. 1, Box 1).

The solution was incubated for 16 hours (FIG. 1, Box 2), after which theresin beads were filtered and rinsed twice with water to separate anyunreacted donor chemical from the acceptor product (FIG. 1, Box 3). Thedonor products were measured using an x-ray fluorescence spectrometerequipped with a 60 watt rhodium anode x-ray tube and a silicon driftdetector (FIG. 1, Box 4). The ratio of the sulfur x-ray fluorescencesignal to the rhodium x-ray fluorescence signal was plotted, and FIG. 3shows the plotted data. Control samples of donor chemical were describedby the equation: (Rh)=0.0706(S)+106.03, where (Rh) is the rhodium x-rayfluorescence signal, (S) is the sulfur x-ray fluorescence signal. Thisdata is labeled as “Control” in FIG. 3. Many of the donor chemical whichhad been subjected to the above reaction conditions had a higher ratioof rhodium x-ray fluorescence signals to sulfur x-ray fluorescencesignal, indicating that the trypsin had removed cysteine₂ with varyingefficiencies. This data is labeled as “Exposed to Protease” in FIG. 3.None of the donor chemical which had been subjected to the abovereaction conditions had sulfur x-ray fluorescence signals of zero,indicating that the trypsin had not removed cysteine₁ from any beadcompletely.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

The embodiment(s) were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. A method for analyzing reactions, comprising: (a)providing a solution comprising solvent, at least one acceptor chemicaland at least one donor chemical, wherein the donor chemical is capableof donating a chemical moiety comprising a chemical element to theacceptor chemical, and at least two controller chemicals capable ofaffecting the reaction between the donor chemical and the acceptorchemical; (b) incubating the solution to allow a portion of the acceptorchemical to react with the donor chemical to form an acceptor product;(c) separating at least a portion of the unreacted donor chemical fromthe acceptor product; (d) removing at least a portion of the solventbefore measuring using x-ray fluorescence at least one of the chemicalsselected from the list of acceptor product and the donor chemical; and(e) measuring using x-ray fluorescence at least one of the acceptorproduct or the donor chemical, wherein: the controller chemicals areenzyme inhibitors; and the sample of donor chemical or acceptor productthat is subjected to x-ray fluorescence measurement is concentrated suchthat the area containing more than 50% of the sample based on the totalamount of the sample of donor chemical or acceptor product that issubjected to x-ray fluorescence measurement is less than 0.005 squarecentimeters.
 2. The method of claim 1, wherein the step of removing atleast a portion of the solvent is performed using reduced pressure. 3.The method of claim 1, wherein at least 80% of the solvent is removed.4. The method of claim 3, wherein the step of separating at least aportion of the unreacted donor chemical from the acceptor product isperformed using chromatography.
 5. The method of claim 3, wherein thestep of separating at least a portion of the unreacted donor chemicalfrom the acceptor product is performed using a centrifuge.
 6. The methodof claim 3, wherein the step of separating at least a portion of theunreacted donor chemical from the acceptor product is performed usingsize exclusion chromatography.
 7. The method of claim 1, wherein thereaction between the donor chemical and the acceptor chemical forms atleast one chemical bond.
 8. The method of claim 1, wherein the reactionbetween the donor chemical and the acceptor chemical breaks at least onebond.
 9. The method of claim 1, wherein the enzyme inhibitor is selectedfrom the group consisting ofphthalene-1-sulfonyl-1H-hexahydro-1,4-diazepine;1-(5-isoquinolinesulfonyl)-1H-hexahydro-1,4-diazepine;1-(5-isoquinolinesulfonyl)-2-methylpiperazine;1-(5isoquinolinesulfonyl)-piperazine; 1,2-Dimethoxy-N-methyl(1,3)benzodioxolo(5,6-c) phenanthridinium chloride;1-[N,0-bis-(5isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one;2,3-Dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide; 2,5-Dihydroxymethylcinnamate;2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide;3-[[4(dimethylamino)phenyl]methylene]-1,3-dihydro-2H-indol-2-one;4-(2,5-Dihydroxy-benzylamino)benzoic Acid Adamantan-1-yl Ester;4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3morpholinopropoxy)quinazoline;4,5,6,7-Tetrabromo-2-azabenzimidazole;4-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline;4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,d]pyrimidine;4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;4-Amino-5-iodo-7-(beta-D-ribofuranosyl)pyrrolo[2,3d]-pyrimidine;4′-Amino-6-Hydroxyflavone; 4-Amino-N-(2,5-dihydroxybenzyl)adamantylBenzoate; 4-Amino-N-(2,5-dihydroxybenzyl)methylbenzoate;5,6-Dichloro-1-b-D-ribofuranosylbenzimidazole;5,7-Dimethoxy-3-(4-pyridinyl)quinoline;6-Amino-4-methyl-8-([8-D-ribofuranosyl)4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine;Bisindolylmaleimide IX; N-(2′-Guanidinoethyl)-5-isoquinolinesulfonamide;N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide;N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide;N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide;N-Benzyl-3,4-dihydroxy-enzylidenecyanoacetamide; Picropodophyllin;Quercetin; rac-2-methyl-1-octadecyl-glycero-(3)-phosphocholine;Rapamycin; Rottlerin; Staurosporine;trans-3,3′,4,5′-Tetrahydroxystilbene;4-[(1E)-2-(3,5-Dihydroxyphenyl)ethenyl]-1,2-benzenediol;trans-4-[(1R)-1-Aminoethyl]N4pyridinylcyclohexanecarboxamide andwortmannin.